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Fatty acid‑activated proton transporter SR4 prevents hepatic steatosis and metabolic alterations in diabetic mice by improving mitochondria function, energy balance and oxidative stress

  • Authors:
    • James Figarola
    • Jyotsana Singhal
    • Sharad Singhal
  • View Affiliations / Copyright

    Affiliations: Department of Diabetes Complications and Metabolism, Arthur Riggs Diabetes and Metabolism Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA, Department of Medical Oncology and Therapeutics Research, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA
    Copyright: © Figarola et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 195
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    Published online on: May 20, 2026
       https://doi.org/10.3892/etm.2026.13190
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Abstract

Type 2 diabetes (T2D) is a growing global health crisis, largely driven by rising obesity rates. Untreated T2D leads to severe complications such as cardiovascular disease, nephropathy, retinopathy, neuropathy and hepatic dysfunction. Current therapies primarily manage hyperglycemia but often fail to address core pathophysiological drivers such as insulin resistance and obesity. This highlights an urgent need for novel therapeutics with distinct mechanisms, particularly those targeting energy metabolism and insulin sensitivity, to improve long‑term T2D outcomes. Modulating mitochondrial respiration through mild uncoupling has emerged as a promising strategy to promote negative energy balance. SR4, a small‑molecule mitochondrial uncoupler, represents a novel class of fatty acid‑activated proton transporters. In the present study, the metabolic effects of oral SR4 administration were investigated in male db/db mice, a model of T2D. SR4 significantly reduced body weight gain and improved body composition by selectively decreasing fat mass without affecting lean mass. Indirect calorimetry demonstrated that SR4 treatment increased oxygen consumption and total energy expenditure, independent of food intake. Importantly, SR4 notably improved glycemic control, reduced insulin resistance and prevented dyslipidemia, hepatic steatosis and liver injury. Mechanistically, SR4 activated hepatic AMPK, enhanced mitochondrial respiration and mitigated oxidative stress. Liver transcriptomic profiling further demonstrated broad metabolic reprogramming, including downregulation of lipogenesis and PPARγ signaling, concurrently with the upregulation of genes involved in energy metabolism and antioxidant defense. Collectively, these findings demonstrated that SR4 ameliorates multiple aspects of metabolic dysfunction in an obese T2D mouse model by targeting key pathways in energy regulation and lipid metabolism. The present results provided additional mechanistic insights into the effects of mitochondrial uncouplers in the liver and support further investigation of SR4 and related fatty acid anion transporters as a novel therapeutic class for metabolic diseases.
View Figures

Figure 1

SR4 reduces BW and body fat mass
without altering food intake in db/db mice. (A)
Representative images of mice from each treatment group,
demonstrating differences in overall body shape. (B) Weekly mean BW
of vehicle vs. SR4-treated mice. (C) Quantification of body fat
mass and lean mass percentage after 5 weeks of treatment,
determined using EchoMRI™ technology. (D) Mean food
intake of each mouse was manually measured. All quantitative data
are presented as the mean ± SEM, with n=8 animals per group.
Statistical significance is indicated as follows:
**P<0.01 and ***P<0.001 for SR4 vs.
control. BW, body weight.

Figure 2

SR4 increases oxygen consumption and
energy expenditure in db/db mice. (A) Daily total oxygen
consumption rates, (B) energy expenditure and (C) RER of control
and SR4-treated mice. (D) Average oxygen consumption, (E) energy
expenditure and (F) RER in both light and dark cycles. All
quantitative data are presented as the mean ± SEM, with n=8 animals
per group. Statistical significance is indicated as follows:
**P<0.01 for SR4 vs. control. RER, respiratory
exchange ratio; VO2, maximal oxygen consumption.

Figure 3

SR4 improves glycemic control in
db/db mice. (A) Fasting plasma glucose, (B) HbA1c, (C)
fasting plasma insulin, (D) intraperitoneal glucose tolerance test
and (E) total AUC. All quantitative data are presented as the mean
± SEM, with n=8 animals per group. Statistical significance is
indicated as follows: **P<0.01 and
***P<0.001 for SR4 vs. control. HbA1c, glycated
hemoglobin; AUC, area under the curve.

Figure 4

SR4 ameliorates dyslipidemia and
hepatic steatosis in db/db mice. Plasma (A) TG and (B)
cholesterol levels, (C) liver weight and liver TG content (D) of
control and SR4-treated mice at the end of the present study. (E)
Representative images of liver sections stained with H&E and
Oil Red O. Magnification, x160, scale bar 100 µm. (F) Quantitative
PCR analysis showing the relative mRNA expression levels of lipid
metabolism-associated genes Acaca, Acly,
Cebpa, Fasn, Pparg, Scd1 and
Srepbf1 in liver of mice with and without SR4 treatment.
Plasma levels of liver injury enzymes (G) ALT and (H) AST. All
quantitative data are presented as the mean ± SEM, with n=8 animals
per group for panels A-D and G-H and n=4 animals per group for
panel F. Statistical significance is indicated as follows:
*P<0.05, **P<0.01 and
***P<0.001 for SR4 vs. control. ALT, alanine
transaminase; AST, aspartate aminotransferase; Acaca,
acetyl-coenzyme A carboxylase; Acly, ATP citrate lyase;
Cebpa, CCAAT/enhancer-binding protein a; Fasn, fatty
acid synthase; Pparg, peroxisome proliferator-activated
receptor g; Scd1, stearoyl-coenzyme A desaturase 1;
Srebf1, sterol regulatory element binding protein-1c; TG,
triglycerides.

Figure 5

SR4 activates hepatic AMPK, increases
liver mitochondrial respiration and alleviates oxidative stress in
db/db mice. (A) Liver AMP:ATP ratio in control and
SR4-treated animals (n=5). (B) Representative western blotting
analysis of protein lysates from liver (n = 3). Lysates were
subjected to SDS-PAGE and immuno-blotted with antibodies specific
to phosphorylated and total AMPK, phosphorylated and total ACC or
β-actin. (C) Seahorse analysis of mitochondrial function and
bioenergetics of freshly isolated liver mitochondria (n=4-5 per
group) after 5 weeks of SR4 treatment. Real time OCR and key
respiration parameters such as (D) basal respiration (state 2),
ADP-linked respiration (state 3), maximal respiration (state 3u),
proton leak (state 4) and spare respiratory capacity are shown. (E)
Hepatic levels of MDA, measured as TBARS and antioxidants (F) GSH,
(G) GPx and (H) GST in liver lysates of control and SR4-treated
mice (n=3). Statistical significance is indicated as follows:
*P<0.05, **P<0.01 and
***P<0.001 for SR4 vs. control. p-, phosphorylated.
ACC, acetyl-CoA carboxylase; OCR, oxygen consumption rate; MDA,
malondialdehyde; FCCP, carbonyl
cyanide-p-trifluoromethoxyphenylhydrazone; GSH, glutathione; GST,
glutathione S-transferase; GPx, glutathione peroxidase; TBARS,
thiobarbituric acid reactive substances.

Figure 6

Transcriptomic analysis of hepatic
gene expression in SR4-treated db/db mice. (A) Volcano plot
of DEGs between SR4 and vehicle-treated db/db mice. DEGs
with P<0.05 and a fold-change ≥1.5 are depicted in red
(downregulated) and blue (upregulated) circles. Black circles
represent DEGs below the cut-off. Annotated dots correspond to the
top 10 DEGs with the largest (Manhattan) distance from the origin
above the significance thresholds indicated by the dashed line.
Data plotted using the VolcaNoseR web tool. (B) Bar plots of the
top ten significantly enriched upregulated (left panel) and
downregulated (right panel) GO biological processes. (C) Bar plots
of the top ten significantly enriched upregulated (left panel) and
downregulated (right panel) KEGG pathways. GO, gene ontology; KEGG,
Kyoto Encyclopedia of Genes and Genomes.

Figure 7

PPI networks and subnetworks
(modules) of DEGs in SR4-treated db/db mice livers. (A) PPI
network of all 642 DEGs. For clarity, disconnected nodes are not
shown. Circles represent genes (nodes) and lines between them
represent interactions (edges). The top 5 most significant modules
from the PPI network of DEGs, identified by MCODE screening. These
modules are associated with (B) steroid hormone biosynthesis, (C)
pentose phosphate pathway and glycolysis, (D) fatty acid synthesis
and pyruvate metabolism, (E) lipid droplet metabolism and (F)
metabolism of amino acids and derivatives. (G) PPI subnetwork for
genes associated with AMPK and nutrient and energy metabolic
pathways, as identified by GO and KEGG enrichment analyses. (H) Top
10 hub genes identified in the subnetwork in (G) using the ‘Maximal
Clique Centrality’ plugin of CytoHubba. (I) Putative
AMPK/PPARγ-associated regulatory axis for fatty acid and TG
synthesis, TG production, lipid droplet formation and storage. In
panels (A-G) and (I), green and red circles represent upregulated
and downregulated genes, respectively. In panel (H), red circles
indicate hub genes with high-ranking scores, while yellow
rectangles represent hub genes with lower-ranking scores. PPARγ,
peroxisome proliferator-activated receptor γ; PPI, protein-protein
interaction; TG, triglycerides; DEGs, differentially expressed
genes.

Figure 8

Summary of the metabolic effects of
SR4 on the liver of db/db mice. SR4 functions as a
mitochondrial uncoupler by facilitating fatty acid-activated proton
transport across the inner mitochondrial membrane. As a bisaryl
urea-based anion transporter, SR4 enhances the flip-flop movement
of deprotonated fatty acids across the inner mitochondrial
membrane, promoting proton leak. This uncoupling disrupts the
mitochondrial proton gradient, leading to reduced ATP synthesis.
The resulting decline in cellular ATP levels activates AMPK, a key
regulator of energy homeostasis. In the liver, AMPK activation
suppresses lipogenic pathways, including PPARγ signaling, while
promoting catabolic processes that support mitochondrial function
and energy utilization. This metabolic reprogramming enhances
mitochondrial respiration and oxidative capacity, reduces oxidative
stress and improves metabolic flexibility. Collectively, these
effects contribute to broad metabolic improvements in db/db
mice, including increased energy expenditure and metabolic
activity, reduced nody weight, improved glycemic control, enhanced
insulin sensitivity, correction of dyslipidemia and attenuation of
hepatic steatosis and injury. Figure partly created in BioRender.
PPARγ, peroxisome proliferator-activated receptor γ.
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Copy and paste a formatted citation
Spandidos Publications style
Figarola J, Singhal J and Singhal S: Fatty acid‑activated proton transporter SR4 prevents hepatic steatosis and metabolic alterations in diabetic mice by improving mitochondria function, energy balance and oxidative stress. Exp Ther Med 32: 195, 2026.
APA
Figarola, J., Singhal, J., & Singhal, S. (2026). Fatty acid‑activated proton transporter SR4 prevents hepatic steatosis and metabolic alterations in diabetic mice by improving mitochondria function, energy balance and oxidative stress. Experimental and Therapeutic Medicine, 32, 195. https://doi.org/10.3892/etm.2026.13190
MLA
Figarola, J., Singhal, J., Singhal, S."Fatty acid‑activated proton transporter SR4 prevents hepatic steatosis and metabolic alterations in diabetic mice by improving mitochondria function, energy balance and oxidative stress". Experimental and Therapeutic Medicine 32.1 (2026): 195.
Chicago
Figarola, J., Singhal, J., Singhal, S."Fatty acid‑activated proton transporter SR4 prevents hepatic steatosis and metabolic alterations in diabetic mice by improving mitochondria function, energy balance and oxidative stress". Experimental and Therapeutic Medicine 32, no. 1 (2026): 195. https://doi.org/10.3892/etm.2026.13190
Copy and paste a formatted citation
x
Spandidos Publications style
Figarola J, Singhal J and Singhal S: Fatty acid‑activated proton transporter SR4 prevents hepatic steatosis and metabolic alterations in diabetic mice by improving mitochondria function, energy balance and oxidative stress. Exp Ther Med 32: 195, 2026.
APA
Figarola, J., Singhal, J., & Singhal, S. (2026). Fatty acid‑activated proton transporter SR4 prevents hepatic steatosis and metabolic alterations in diabetic mice by improving mitochondria function, energy balance and oxidative stress. Experimental and Therapeutic Medicine, 32, 195. https://doi.org/10.3892/etm.2026.13190
MLA
Figarola, J., Singhal, J., Singhal, S."Fatty acid‑activated proton transporter SR4 prevents hepatic steatosis and metabolic alterations in diabetic mice by improving mitochondria function, energy balance and oxidative stress". Experimental and Therapeutic Medicine 32.1 (2026): 195.
Chicago
Figarola, J., Singhal, J., Singhal, S."Fatty acid‑activated proton transporter SR4 prevents hepatic steatosis and metabolic alterations in diabetic mice by improving mitochondria function, energy balance and oxidative stress". Experimental and Therapeutic Medicine 32, no. 1 (2026): 195. https://doi.org/10.3892/etm.2026.13190
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